ORIGINAL Veterinary ARTICLE Veterinary Research Forum. 2013; 4 (2) 123 - 127 Research Forum Journal Homepage: vrf.iranjournals.ir

Chemical composition and in vitro antibacterial activity of and methanol extract of platyloba D.C against some of food-borne pathogenic bacteria

Mohammad Hashemi1, Ali Ehsani*1, Nima Hosseini Jazani2, Javad Aliakbarlu1, Razzaqh Mahmoudi3

1 Department of Food Hygiene and Quality Control, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran; 2 Food and Beverages Safety Research Center, Urmia University of Medical Sciences, Urmia, Iran; 3 Department of Food Hygiene and Aquatics, Faculty of Veterinary Medicine, University of Tabriz, Tabriz, Iran.

Article Info Abstract Article history: Echinophora Platyloba D.C as a medicinal is used for preservation of foods and treatment of many diseases in different regions of Iran. The present study was undertaken to Received: 26 September 2012 determine the chemical composition and investigation of the antibacterial effects of essential Accepted: 05 January 2013 oil as well as methanol extract from aerial part of Echinophora Platyloba D.C against S. aureus, Available online: 15 June 2013 L. monocytogenes, S. Thyphimurium and E. coli. Chemical analysis using gas chromatography and mass spectrophotometry (GC/MS) showed that ocimene (26.51%), 2,3-Dimethyl-cyclohexa- Key words: 1,3-diene (9.87%), alpha-pinene (7.69%) and gamma-dodecanolactone (5.66%) were dominant components of essential oil and the main constituents of methanol extract were o- Antimicrobial activity Cymene (28.66%), methanol (8.50%), alpha-pinene (7.42%) and gamma-decalactone (5.20%). Echinophora Platyloba D.C The essential oil showed strong antimicrobial activity against tested bacteria, whereas the Essential oil methanol extract almost remained inactive against gram-negative bacteria. The most Methanol extract sensitive bacteria to essential oil and extract of Echinophora Platyloba D.C were L. mono- cytogenes and S. aureus. Minimum inhibitory concentration (MIC) values of essential oil against L. monocytogenes and S. aureus were 6250 and 12500 ppm, respectively. MIC of methanol extract against S. aureus and L. monocytogenes was 25000 ppm. Therefore, purifying and evaluation of antibacterial effects of the active substances of the essential oil and methanol extract of this plant for future application as antibacterial agents and food preservatives to combat pathogenic and toxigenic microorganisms is recommended. © 2013 Urmia University. All rights reserved.

بررسی ترکیبات شیمیایی و خواص ضد باکتریایی اسانس و عصاره متانولی گیاه اکینوفورا پلتی لوبا علیه برخی باکتریهای بیماریزا در شرایط آزمایشگاهی چکیده اکینوفورا پلتی لوبا در نواحی مختلف ایران به عنوان یک گیاه دارویی جهت نگهداری مواد غذایی و درمان بسیاری از بیماری ها استفاده می شود. مطالعه حاضر جهت شناسایی ترکیبات شیمیایی و ارزیابی اثرات ضد باکتریایی اسانس و عصاره متانولی قسمت های هوایی گیاه اکینوفورا پلتی لوبا در مقابل باکتری های استافیلوکوکوس اورئوس، لیستریا مونوسایتوژنز، سالمونال تیفی موریوم و اشریشیا کوالی صورت گرفته است. آنالیز شیمیایی نشان داد که ترکیبات غالب اسانس شامل اسیمن )15/62 درصد(، 6و3 دی متیل- سیکلوهگزا- 5و3 دی ان )78/9 درصد(، آلفا پی ان )29/8 درصد( و گاما- دودکانوالکتون )22/1 درصد( و ترکیبات اصلی عصاره متانولی شامل اسیمن )22/67 درصد(، متانول )15/7 درصد(، آلفا پی ان )26/8 درصد( و گاما- دودکانوالکتون )65/1 درصد( بودند. اسانس اکینوفورا پلتی لوبا در برابر باکتری های مورد آزمایش اثر ضد باکتریایی نسبتاً قوی داشت در حالیکه عصاره آن در برابر باکتری های گرم منفی تقریباً غیر مؤثر بوده است. باکتری های لیستریا مونوسایتوژنز و استافیلوکوکوس اورئوس حساس ترین باکتری ها در مقابل اسانس و عصاره اکینوفورا پلتی لوبا بوده اند. حداقل میزان بازدارندگی اسانس در برابر لیستریا مونوسایتوژنز و استافیلوکوکوس اورئوس به ترتیب 2615 و ppm 56155 و حداقل میزان بازدارندگی عصاره متانولی در برابر این دو باکتری ppm 61555 می باشد. بر این اساس خالص سازی و ارزیابی اثرات ضد باکتریایی ترکیبات فعال اسانس و عصاره متانولی این گیاه جهت استفاده آن به عنوان ترکیب ضد باکتریایی و نگهدارنده مواد غذایی بر علیه میکروارگانیسم های مسمومیت زا و بیماری زا توصیه می شود.

واژه های کلیدی: اسانس گیاهی، اکینوفورا پلتی لوبا، فعالیت ضد باکتریایی، عصاره متانولی

*Correspondence:

Ali Ehsani, DVM, PhD Department of Food Hygiene and Aquatics, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran. E-mail: [email protected] 124 M. Hashemi et al. Veterinary Research Forum. 2013; 4 (2) 123 - 127

Introduction containers protected completely from light. Preparation of the essential oil. Dry aerial parts Due to the side effects of chemical and synthetic (100 g) of Echinophora Platyloba D.C were subjected to the antimicrobial agents and emerging increase in bacterial hydrodistillation for 2.5 hr, using a clevenger-type resistances to current antibiotics and other antimicrobial apparatus, according to the method recommended by the agents, more studies have been recently focused on European Pharmacopia.13,14 The obtained essential oil was characterization of novel potential natural antimicrobial dried over anhydrous sodium sulphate and stored at 4 ˚C agents from plant, animal and microbial sources.1 for further experiments. are rich in a wide variety of secondary metabolites, such as Preparation of methanol extract. The air-dried and tannins, terpenoids, alkaloids and flavonoids, which have finely ground sample was extracted using the method been reported to have in vitro antimicrobial properties.2 described previously.15 Briefly, 100 g of sample were Essential oils and their components are gaining increasing extracted in a Soxhlet with methanol in 60 ˚C for 6 hr. The interest because of their relatively safe status, their wide extracts were then filtered with Whatman filter paper acceptance by consumers and their exploitation for number 1 and concentrated in vacuo at 45 ˚C by a rotary potential multi-purpose functional use.3 Many researchers evaporator (Heidolph laborta 4003, Schwabach, Germany) have reported the antimicrobial, antifungal and anti- yielding a waxy material. Extracts were concentrated in oxidant properties of essential oils.4-8 vacuo, dried, and kept in the dark at 4 ˚C until tested. A knowledge of the chemical constituents of plants is Analysis of the essential oil and extract. Chemical desirable not only for the discovery of therapeutic agents composition of the essential oil and extract were analyzed but because such information may be of value in disclosing by gas chromatography. The gas chromatograph (Agilent new sources of such economic materials as tannins, oils, 6890, Swindon, UK) was equipped with an HP-5MS gums, precursors for the synthesis of complex chemical capillary column (30 × 0.25 mm ID × 0.25 mm film substances, etc. In addition, the knowledge of the chemical thickness) and the data were taken under the following constituents of plants would further be valuable in conditions: initial temperature 50 °C, temperature ramp 5 °C discovering the actual value of folkloric remedies.5 The per min, 240 °C min to 300 °C (holding for 3 min), and genus Echinophora (Umbelliferae, subfamily Apioideae, injector temperature at 290 °C. The carrier gas was helium tribe Echinophoreae), is represented in the plant flora of and the split ratio was 0.8 mL-1 per min. For confirmation Iran.9 Echinophora Platyloba D.C, a member of the of results, essential oil was also analyzed by gas chromato- Umbelliferae family, is a perennial plant, distributed only in graphy mass spectrometry (Agilent 6890 gas chromatograph Mediterranean region mostly at maritime sands also equipped with an Agilent 5973 mass-selective detector; which could be found in some central and western Agilent, Swindon, UK) and the same capillary column and provinces of Iran.6,10 The underground rhizome has a wide analytical conditions were used as mentioned above. The growth and the erected stem is full of branches. Leaves MS was run in electron-ionization mode with ionization end with spines. It blossoms from June to September. In energy of 70 eV (Wiley-VCH 2001, Weinheim, Germany). Iran, fresh and dried aerial parts of some of these species Bacterial strains. The essential oil and extract were are added to cheese and yoghurt for flavoring, the species individually tested against two gram-negative (S. of the Echinophora genera are also used in folk medicine to Thyphimurium ATCC 13311 and E. coli ATCC 43894) and heal wounds and to treat gastric ulcers due to its anti- two gram-positive bacteria (S. aureus ATCC 6538, L. fungal, carminative, and digestive properties,11 as a stimulant monocytogenes ATCC 19118). Lyophilized cultures of the and an invigorator of the stomach and its anti-microbial organisms were obtained from the culture collection of the and anti-cancer effects have been shown, recently.12 Department of Microbiology, Faculty of Veterinary The objectives of this study were 1) to determine the Medicine, University of Tehran, Tehran, Iran. chemical composition (based on GC-MS results) of hydro- Micro-well dilution assay. The minimum inhibitory distilled essential oils and methanol extract of Echinophora concentration (MIC) and minimum bactericidal concentration Platyloba D.C and 2) to investigate their antibacterial activities. (MBC) values of the essential oil and extract were studied for the bacterial strains in micro plates. The inocula of the Materials and Methods bacterial strains were prepared from 18 hr nutrient broth cultures and suspensions were adjusted to 0.5 McFarland Plant material. The aerial parts of Echinophora standard turbidity. Essential oil and extract were dissolved Platyloba D.C was collected during flowering stage (10th in 10% dimethyl sulfoxide. Then, the solution firstly was June to 15th August 2010) from Maragheh city, northwest diluted to the highest concentration (100000 ppm) as a of Iran and identified by the Herbarium of West Azerbaijan stock solution, and then serial two-fold dilutions were Agricultural and Natural Resource Center, Urmia, Iran made in a concentration range from 1562.5 to 100000 ppm (Voucher specimen no. : 6502). Then, it was dried and in nutrient broth. Minimum inhibitory concentration ground into powder. The prepared powder was kept in tight values of essential oil and extract against bacterial strains

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were determined based on a micro-well dilution method. 6250 and 12500 ppm, respectively. The minimum The 96-well plates were prepared dispensing 160 µL of inhibitory concentration of extract against S. aureus and L. nutrient broth and 20 µL of the inoculums into each well. monocytogenes was 25000 ppm. A 20 µL aliquot from the stock solutions of essential oil Table 1. Chemical composition (%) of Echinophora Platyloba D.C initially prepared at the concentration of 100000 ppm essential oil analyzed by GC/MS. were added into the first wells. Then, 20 µL from their Compound KI Composition serial dilutions was transferred into consecutive wells. Hexanal 806 1.25 The last well containing 180 µL of nutrient broth without 2,3 -Dimethyl-cyclohexa-1,3-diene 863 9.87 any chemical compound and 20 µL of the inoculum on Alpha -pinene 948 7.69 each strip which were used as the negative control. The Ocimene 958 26.51 final volume in each well was 200 µL.16 The plates were Beta -linalool 1082 1.80 covered with a sterile plate sealer. Contents of each well Benzopyran 1342 1.18 were mixed on plate shaker at 300 rpm for 20 sec and Cyclohexene, 2-ethenyl-1,3,3-trimethyl 1105 1.44 2,5-Octadecadiynoic acid, methyl ester 2112 2.30 then incubated at appropriate temperatures for 24 hr. Caryophyllene 1494 2.48 Microbial growth was determined by absorbance at 600 nm Dihydropseudoionone 1420 1.48 using the ELx 800 universal micro-plate reader (Biotek Gamma -dodecanolactone 1582 5.84 Instrument Inc., Winooski, VT, USA) and confirmed by 4 -(2,2-Dimethyl-6-methylenecyclohexylidene) 1475 1.13 plating 5 µL samples from clear wells on nutrient agar - 3-methylbutan-2-1 medium. The MIC and MBC were defined as the lowest Nerolidol 1564 5.66 concentration of the compounds to inhibit the growth of All -trans-farnesol 1710 3.30 microorganisms and show bactericidal effects on micro- Gamma -dodecalactone 1582 3.28 Heptacosane 2705 2.10 17,18 organisms, respectively. Nonacosane 2904 4.11 Statistical analysis. All data were expressed as Cis -Z-alpha-bisabolene epoxide 1531 1.11 mean ± standard deviations (SD) of triplicate measure- 2 -[4-6-(2,6,6-trimethylcyclohex-1-enyl)hexa- 2561 2.89 ments. Data analysis was performed using Graphpad 1,3,5 -trienyl]cyclohex-1-en-1-carboxaldehyde Prism (Version 5.0 for Windows, Graphpad software, Sum - 85.42 San Diego, CA, USA). Discussion Results The oil composition of different species from The yield of essential oil and methanol extract were 0.3% Echinophora genus has been studied and various and 1.5% (v/W) based on dry weight, respectively. The compounds such as 3-carene,9 (E)-β-ocimene,19 a- chemical compositions of E. platyloba D.C essential oil and phellandrene20 and delta-3-carene21 have been reported as extract are summarized in Tables 1 and 2. Ninety and 21 the first major constituent. Rahimi-Nasrabadi et al. compounds were characterized representing 85.42% and indicated that major essential oil composition of the aerial 91.68% of the total content of essential oil and methanol parts of Echinophora platyloba growing wild in Isfahan extract, respectively. province of Iran is bata-ocimene, delta-3-carene and The major compound of essential oil was ocimene limonene which are quite consistent with the results of (26.51%), following by 2,3 Dimethyl-1,3-cyclohexadiene chemical analysis of current study.19 (9.87%), alphapinene (7.96%), gamma-dodecalactone Most studies concerning the antimicrobial mode of (5.84%) and nerolidol (5.66%). Ocimene (28.66%) was action of essential oil constituents have been performed on the most abundant components in E. platyloba D.C bacteria, gram-negative bacteria are generally less methanol extract and the other major components were susceptible than gram-positive bacteria.22 The outer beta-cis-ocimene (9.77%), methanol (8.57%), alpha- membrane of gram-negative bacteria contains hydrophilic pinene (7.42%), gamma.-decalactone (5.20%) and beta- lipopolysaccharides (LPS), which create a barrier toward linalool (4.99%). The minimum inhibitory concentration macromolecules and hydrophobic compounds, providing and MBC values of E. platyloba D.C essential oil and extract gram-negative bacteria with higher tolerance toward on different kinds of bacteria in this study are summarized hydrophobic antimicrobial compounds like those found in in Tables 3 and 4. The essential oil showed strong essential oils.23 Minimum inhibitory concentration and antimicrobial activity against tested bacteria, whereas the MBC values of E. platyloba D.C essential oil on different methanol extract almost remained inactive against gram- kinds of bacteria in this study indicated the notable negative bacteria. The most sensitive bacteria to essential sensitivity of gram-positive bacteria and the relative oil and extract of Echinophora Platyloba D.C were L. susceptibility of gram-negative bacteria. monocytogenes and Staphylococcus aurues, MIC values of An study by Entezari et al. indicated that methanolic essential oil against L. monocytogenes and S. aureus were extract of E. platyloba D.C can inhibit the growth of two

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Table 2. Chemical composition (%) of Echinophora Platyloba D.C Table 4. Antimicrobial properties (MIC, MBC) of Echinophora methanolic extract analyzed by GC/MS. Platyloba D.C methanolic extract. Compound KI Composition Microorganisms MIC (ppm) MBC (ppm)

Methanol 315 8.50 S. aureus 25000 * Alpha-pinene 948 7.42 L. monocytogenes 25000 * Beta-terpinene 1071 1.49 E. coli * * Beta-myrcene 993 3.22 S. typhimurium * * O-cymene 1028 28.66 * No effect was determined while the highest concentration was used. beta.-cis-Ocimene 1040 9.77 Isopinocarveol 1198 1.06 α-terpinene showed no or low antimicrobial activity Beta-Linalool 1109 4.99 13-Tetradece-11-yn-1-ol 1663 1.08 against 25 different genera of bacteria that pose problems 2-Nonenal 1161 1.31 in animals, plants, and food products.26 Koutsoudaki et al. Naphthalene 1157 3.47 compared the antibacterial effects of α-pinene, β-pinene, 5-Isopropenyl-2-methyl-7-oxabicyclo[4.1.0]- 1294 1.42 p-cymene, β-myrcene, β-caryophyllene, limonene and γ- heptan-2-ol terpinene on E. coli, S. aureus and Bacillus cereus, their Acetic acid 1009 1.84 antimicrobial activity were low or absent.27 The p-cymene Trans-Z-alpha-bisabolene epoxide 1746 1.34 5,6,6-Trimethyl-5-(3-oxobut-1-enyl)-1- 1442 1.00 and γ-terpinene were ineffective as fungicides against oxaspiro[2.5]octan-4-1 Saccharomyces cerevisiae.28 These in vitro tests indicate Gamma-decalactone 1431 5.20 that terpenes are inefficient as antimicrobials when Formic acid 1576 1.24 applied as single compounds. Spathulenol 1699 2.92 In conclusion, regarding this study, it is clear that E. Trans-Farnesol 2021 3.17 2-Butyloxycarbonyloxy-1,1,10-trimethyl-6,9- 3942 1.60 platyloba D.C essential oil and its methanol extract indeed epidioxydecalin exhibit antibacterial activity. Its antibacterial activity 1.3-Ethyl-5-(2'-ethylbutyl)octadecane 3430 1.00 against L. monocytogenes was the highest followed by S. Sum - 91.68 aureus and E. coli. Therefore, it can be suggested to purify and evaluate the antibacterial effects of active substances bacterial species of S. aureus and P. aeruginosa.3 In the of E. platyloba D.C essential oil and methanol extract for current study S. aureus was one of the most sensitive therapeutic or industrial utilization. tested bacteria to essential oil and extract of Echinophora Platyloba D.C as well and it’s growth was inhibited in MIC Acknowledgments values of 12500 and 25000 ppm for essential oil and extract, respectively. This work was financially supported by Faculty of Results of this study suggested that E. platyloba D.C Veterinary Medicine, Urmia University and Food and essential oil and extract have potential effects as antimicrobial Beverage Research Center, Urmia University of Medical agents. Main components of essential oil and extract, such Sciences, Urmia, Iran. The authors wish to thank Dr. H. as ocimene, α-pinene, myrcene and α-phellandrene have Hasanzadazar and Mrs. M. Heydari for their technical been previously reported to have antibacterial activity.11 assistance. The carvacrol precursor p-cymene is a monoterpene that has a benzene ring without any functional groups on References its side chains. P-cymene has a high affinity for membranes and causes membrane expansion and affect the membrane 1. Hussain AI, Anwar F, Sherazi STH, et al. Chemical potential of intact cells.24 P-cymene had a negligible effect composition, antioxidant and antimicrobial activities of on the protein synthesis of E. coli cells, while its effect on basil (Ocimum basilicum) essential oils depends on the membrane potential resulted in decreased cell motility, seasonal variations. Food Chem 2008; 108: 986-995. as a proton motive force is needed for flagellar movement.25 2. Cowan MM. Plant products as antimicrobial agent. Clin Terpenes are hydrocarbons produced from combination Microbial 1999; 12: 564-582. of several isopreneunits (C5H8). In a large scale experiment, 3. Entezari M, Hashemi M, Ashki M, et al. Studying the limonene, α-pinene, β-pinene, δ-3-carene, sabinene, and effect Echinophora platyloba extracton bacteria (Staphylococcus aureus and Pseudomonas aeruginosa) Table 3. Antimicrobial properties (MIC and MBC) of Echinophora Platyloba D.C essential oil. and fungi (Candida albicans, Aspergillus flavus and Microorganisms MIC (ppm) MBC (ppm) Aspergillus niger) in vitro. World J Med Sci 2009; 4: S. aureus 12500 25000 89-92. L. monocytogenes 6250 6250 4. Scchetti G, Maietti S, Muzzoli M, et al. Comparative E. coli 50000 * evaluation of 11 essential oils of different origin as S. typhimurium * * functional antioxidants, antiradicals and antimicrobials * No effect was determined while the highest concentration was used. in foods. Food Chem 2005; 91: 621-632.

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